This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The importance of ECM mechanics on 2-D cell responses (e.g., adhesion, spreading, motility, proliferation, and even differentiation) is widely recognized and well characterized. However, the effects of intrinsic mechanical cues on longer-term phenotypic responses of cells in 3-D culture remain undefined, as do the molecular mechanisms underlying these phenotypic changes. Efforts to relate changes in cell phenotype with substrate mechanics in 3-D have been hindered in part by the lack of suitable material systems. Ideally, a suitable material system should provide the means to predictably tune substrate mechanical properties independently from adhesion ligand density and proteolytic sensitivity. In addition to material limitations, efforts to dissect the influence of ECM mechanics on cell function in 3-D have been hampered by the lack of suitable methods to assess mechanical properties at the local cell-material interface. Most researchers have instead chosen to utilize bulk measurements of a material's elastic and viscoelastic properties and to correlate these with cell function;unfortunately, these do not adequately depict the local microenvironment. We proposed to utilize a unique biosynthetic hybrid hydrogel based on poly(ethylene glycol) and fibrinogen. Furthermore, we proposed to develop novel methodologies to measure the local mechanical properties this material. The following three specific aims constitute the proposed study.